The absence of clear immune correlates for protection against HIV-1 highlight the critical need to identify new pathways of host-resistance from infection. The overall goal of this R21 proposal is to identify novel mechanism(s) of protection in a cohort of HIV-exposed individuals who remain sero-negative (HESN) despite many years of high-risk behavior and exposure. Previous studies of HESN subjects exposed to HIV-1 through IV-drug use and needle-sharing (HESN-IDU) have identified several potential innate and intrinsic mechanisms of protection, including heightened Natural Killer (NK) cell function and increased resistance of CD4+ T cells to HIV-1 infection. Our preliminary data now provide the basis to test an innovative model for how these innate and intrinsic mechanisms of resistance may cooperate to provide a sustained barrier against HIV-1 infection in HESN-IDU subjects. Using a well-described cohort of high-risk HESN-IDU subjects from Philadelphia, we have identified a unique proteomic signature on NK cells from HESN-IDU subjects including the elevated expression of multiple interferon-induced proteins such as ISG-15, MHC-Class I, Granzyme, STAT1/2, as well as the increased expression of several members of the S100 family of immuno-modulatory proteins not previously identified in HESN subjects. Specifically, we identified the increased expression of two cytoplasmic S100 proteins, S100A4 and S100A6, that may stimulate NK degranulation capacity against virally infected cells and a secreted S100 protein, S100A14, that may augment HIV-1 resistance in CD4+ T cells. These results indicate that high-risk needle sharing in protected HESN-IDU subjects may trigger an anti-viral environment involving secreted Interferon and/or S100 proteins that can lead to greater NK activity against virally infected cells and increased CD4+ target cell resistance to HIV-1. In Specific Aim 1, we will measure the ability of NK cells from HESN-IDU subjects and NS-IDU controls to limit the replication capacity of Autologous HIV-1 infected CD4+ primary T cells using an HIV Suppression Assay. We will also investigate if NK cells from HESN-IDU subjects possess increased CD107a degranulation against HIV-1 infected heterologous SupT1 cells and if this correlates with enhanced S100A4 and S100A6 recruitment into the immunological synapse. In Specific Aim 2, we will test the resistance of purified CD4+ T cells from HESN-IDU subjects to HIV-1 infection and investigate the ability of the secreted S100A14 protein to further limit HIV-1 replication capacity. We will investigate the expression of known and uncharacterized host restriction factors in CD4+ T cells from HESN-IDU subjects by proteome analysis and correlate them with infectivity. Together, the Specific Aims proposed in this R21 will test the novel hypothesis that increased expression of interferon-induced factors and S100 proteins in HESN-IDU subjects augment innate and intrinsic mechanisms of resistance by increasing NK-mediated clearance of virally infected cells and reducing the efficiency of HIV-1 replication in CD4+ T cells.